1. Field of the Invention
This invention relates to mutants of human p53 and methods of identifying mutants of p53. Specifically, the invention relates to isolated polypeptides containing human p53 mutations and to isolated nucleic acids encoding the polypeptides. The invention further relates to the methods of detecting human p53 mutations that are toxic, supertransactivating or tox-suppressor mutations as well as to the identification of compounds, agents or interactive factors, such as peptides, that mimic the toxic or the supertransactivating mutations.
2. Background Art
The most commonly inactivated gene target associated with neoplastic transformation is the tumor suppressor gene p53, a key regulator of cellular mechanisms that maintain genome integrity (Prives and Hall). Normally, cellular stresses including DNA damage, hypoxia, suboptimal growth conditions, nucleotide pool unbalance, and activated oncogenes can induce signaling cascades that converge at p53 and result in greater stability of the protein, primarily through post-translational modifications (Giacca and Kastan; Meek). In this process, the p53 half life is increased due to inhibition of the normal MDM2 associated degradation pathway (Freedman and Levine) leading to greater nuclear retention and also through a coordinated regulation of the stability of the p53 tetramer (Stommel). This produces a rapid, transient increase in nuclear levels of active p53 which causes either growth arrest or apoptosis.
The p53 mediated stress responses occur primarily through sequence-specific transcriptional activation (Prives and Hall) involving several p53 functional domains (Ko and Prives). Beginning at the N-terminus of p53, the first 43 amino acids correspond to an acidic region that contains a major transactivation domain; the region between amino acid 43 and 73 represents a second transactivation domain (Venot et al a). The next region (up to amino acid 92) is proline rich and may be involved in apoptosis induction (Venot et al b) and this is followed by a large DNA binding domain (amino acids. 100 to 300) responsible for sequence-specific DNA recognition. The remaining carboxy terminal portion codes for the nuclear localization and export signals, the oligomerization domain, and a basic regulatory region. The p53 monomer recognizes a pentameric DNA sequence (consensus 5′-RRRCW-3′), and a complete binding site consists of two closely spaced head-to-head pentamers (McLure and Lee, Cho et al.,). The transcriptionally active form of the protein is a tetramer.
Several downstream p53 activated genes (reviewed in Ko and Prives) are associated with the induction of programmed cell death (e.g. bax, IGF-BP3, PIG3), regulation of cell cycle through induction of arrest at the G1/S or G2/M checkpoints in response to DNA damage (e.g. p21, GADD45, cyclin G), and modification of p53 stability/activity (MDM2). The number of p53 controlled genes based on a search for putative p53 binding site in the human genome is quite large (>30 genes) (Tokino et al.,). The differential affinity of p53 for different binding sites provides variability in activation of downstream genes (Resnick L. e al, Wang and Prives) and explains why some p53 mutants found in tumors retain partial p53 response (Ludvig et al).
Besides transcriptional activation, p53 can affect gene expression through physical interactions with TATA binding protein and associated factors (Ko and Prives, Oren fos). Other protein-protein interactions are also likely to play an important role in mediating the p53 response. While the number of proteins interacting with p53 is high, the physiological role of these interactions in many cases is unknown (Prives and Hall). In addition p53 may play a direct role in DNA metabolism as evidenced by nuclease activity and p53 binding to recombinant-like structures (Deppert; Griffith).
Loss of p53 function is highly selected during tumor development as evidenced by p53 mutations in nearly 50% of human tumors (Grennblatt). Most alterations result from single missense mutations in the DNA binding domain that prevent or reduce DNA binding and they generally occur at the most p53 invariant residues (Walker et al). These residues usually directly contact DNA or affect the correct folding and stability of the large DNA binding domain (Cho). The strong selection for missense mutations is probably due to dominant-negative interactions with the wild type protein to generate partially inactive heterotetramers, and possibly to gain of function (Gualberto et al.,). In addition, tumor cells generally accumulate high levels of p53 mutant proteins in the nucleus. This is because there is a system for negative feedback control of p53 levels, mediated by MDM2. Loss of p53 function leads to decreased MDM2 gene transcription and accumulation of mutant p53. Functional p53 inactivation can also be achieved by sequestering the protein outside the nucleus, decreasing its stability, or by mutation in genes involved in the upstream or downstream pathways.
In the present invention, several previously identified p53 functional mutants were placed under the control of the galactose inducible GAL1 promoter, whose expression can be variably regulated. Growth inhibition was generally directly correlated with transactivation proficiency in that over-expression of wild type p53 caused severe reduction in colony size while transactivation mutants had a minor impact on growth. Truncated p53, which cannot form tetramers, caused no apparent growth delay while a mutant retaining partial activity had an impact on growth similar to normal p53. Based on this it is expected that certain p53 alleles can exhibit a stronger effect on growth than the wild type, possibly leading to inviability. This might be due to increased DNA binding affinity, altered specificity, increased stability, a shift toward the tetrameric form, or even stronger protein::protein interactions. Such alleles are expected to function more effectively than wild type p53 in mammalian cells as well. This invention has identified such a class of mutants in yeast and novel p53 alleles that cause lethality in yeast and growth suppression in a human tumor cell line. This invention further provides a screening system that can be used to isolate a variety of toxic or novel p53 alleles in yeast that are normally not detectable and these may prove useful in developing new therapeutic approaches.
The invention provides a novel screen in yeast that allows the identification of p53 alleles exhibiting increased transcriptional activation compared to the wild type. p53 alleles showing supertransactivating transcriptional activity are also provided. The invention therefore allows one skilled in the art to tailor p53 functional control for the various promoters recognized by p53 in terms of strength of induction by p53 (from no induction to supertransactivation) and for combinations of p53 responsive genes (i.e. some genes turned on, some genes turned off). This allows the skilled artisan to change the p53 responsive pathways and therefore the biological outcomes in response to environmental challenges. Supertransactivating mutants expected to represent a better choice in p53 gene therapy approaches because they can retain biological activity in the presence of highly expressed endogenous p53 tumor mutants